表达系统：E.coli
单位定义：
PNGase F: 一个单位定义为10μL的反应体系中，37°C反应1小时从10μg变性RNase B中除去超过95%的碳水化合物所需要的酶量。
O-糖苷酶：一个单位定义为在37°C下，1小时内，100μl反应体系中，从5mg唾液酸酶处理的非变性胎球蛋白中去除0.68nmol O-连接糖所需要的酶量。
唾液酸酶（α2-3,6,8,9）：一个单位定义为在37°C，5分钟内，在10μl的总反应体积中，从1nmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-coumarin (AMC)中切割酶>95%的末端α-Neu5Ac所需的酶量。
β1-4半乳糖苷酶：一个单位定义为10μl反应体系中，37°C条件下，1小时内将1nmol Galβ1-4GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-coumarin中>95%的末端β-D-甘露糖水解所需要的酶量。
β-N-乙酰氨基己糖苷酶：一个单位定义为10μL的反应体系中，37°C，1小时内水解1nmol GalNAcβ1-4Galβ1-4Glc-7-amino-4-methyl-coumarin（AMC）中超过95%的末端β-D-N-乙酰氨基半乳糖所需要的酶量。
本产品是5种E.coli表达的重组酶的混合物， 通过协同作用，可去除糖蛋白中绝大部分N-连接、简单O-连接以及一些复杂O-连接糖链。 具体如下：
①PNGase F：来源于Elizabethkingia miricola，分子量约36KDa，可以选择性水解蛋白质的Asn相连的除含α1-3岩藻糖苷键外所有的N-糖键，切割位点为Asn和最内侧的GlcNAc之间的糖苷键，使得糖蛋白释放以天冬酰胺连接的糖分子，并使得天冬酰胺转化为天冬氨酸，切割的糖型包括高甘露糖、杂合和复杂寡糖。
②O-糖苷酶：即内切-α-N-乙酰氨基半乳糖苷酶，来源于Enterococcus faecalis，分子量约147KDa，在用唾液酸酶去除糖蛋白末端的唾液酸后，O-糖苷酶可催化去除糖蛋白上核心1和核心3结构的O-连接二糖。
③唾液酸酶（α2-3,6,8,9）：来源于Arthrobacter ureafaciens，分子量约100KDa，该酶可以水解糖蛋白和寡聚糖上通过α2-3，α2-6，α2-8和α2-9键连接的唾液酸，其水解α2-3和α2-6键的速度要大于α2-8和α2-9键。
④β1-4半乳糖苷酶：来源于Streptococcus pneumoniae，分子量约250KDa，是一种具有高度特异性的糖苷外切酶，催化水解寡糖末端的非还原的半乳糖残基。
⑤β-N-乙酰氨基己糖苷酶：来源于Streptomyces plicatus，分子量约100KDa，可催化水解寡糖末端β-D-N-乙酰氨基半乳糖及氨基葡糖残基，可用于去除O-GlcNAc（丝氨酸/苏氨酸上的N-乙酰氨基葡萄糖苷酶）。
酶活定义
① PNGase F：一个单位定义为10μL的反应体系中，37°C反应1小时从10μg变性RNase B中除去超过95%的碳水化合物所需要的酶量。
② O-糖苷酶：一个单位定义为在37°C下，1小时内，100μl反应体系中，从5mg唾液酸酶处理的非变性脂球蛋白中去除0.68mmol O-连接糖所需要的酶量。
③ 唾液酸酶（α2-3,6,8,9）：一个单位定义为在37°C，5分钟内，在10μl的总反应体积中，从1mmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-coumarin（AMC）中切割解>95%的末端α-Neu5Ac所需的酶量。
④ β1-4半乳糖苷酶：一个单位定义为10μl反应体系中，37°C条件下，1小时内将1mmol Galβ1-4GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-coumarin中>95%的末端β-D-甘露糖水解所需要的酶量。
⑤ β-N-乙酰氨基己糖苷酶：一个单位定义为10μL的反应体系中，37°C，1小时内水解1mmol GalNAcβ1-4Galβ1-4Glc-7-amino-4-methyl-coumarin（AMC）中超过95%的末端β-D-N-乙酰氨基半乳糖所需要的酶量。
本品含量
| 酶 | 5rxns | 20rxns
| | PNGase F | 2.5KU/支 | 10KU/支 | | O-糖苷酶 | 20KU/支 | 80KU/支 | | α2-3,6,8,9 唾液酸酶 | 100U/支 | 400U/支 | | β1-4 半乳糖苷酶 | 250U/支 | 1000U/支 | | β-N-乙酰氨基己糖苷酶 | 75U/支 | 300U/支 |
|
组分表
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 5rxns | | 20rxns | 储存 | | P1527889WT | 蛋白脱糖混合液 II(2-8℃组分) | P1527889A | 蛋白脱糖混合液 II | 液体 | 25µl | | 100µl | 2-8℃. | | P1527889DT | 蛋白脱糖混合液 II (-20℃组分) | P1527889B | 10×糖苷酶切反应液 | 液体 | 200µl | | 200µl | -20℃ | | | P1527889C | 10% NP-40 | 液体 | 200μl | | 200μl | -20℃ | | | P1527889D | 胎球蛋白A (8mg/ml) | 液体 | 50μl | | 50μl | -20℃ | | | P1527889E | 10×糖蛋白变性缓冲液 | 液体 | 200μl | | 200μl | -20℃ |
注：发货时各组分将按照对应储存温度（2-8°C/-20°C）独立包装，采用差异化温控运输，保障产品最佳性能。若同一订单包含多种温区组分，包裹可能分批次送达，请以实际收货情况为准。收货后，请立即依据组分表标注的温度条件分类存放。所有组分在未拆封、运输及储存条件均符合要求的情况下，有效期为自生产之日起 12 个月。 |
产品应用
去除糖蛋白中绝大部分N-连接、简单O-连接以及一些复杂O-连接糖链。
使用方法（仅供参考）
非变性酶切
① 照以下体系进行反应
| 组分 | 体积 |
|---|
| 底物 | 10-100μg 糖蛋白（10-40μl） | | 10×糖苷酶切反应液 | 5μl | | 蛋白脱糖混合液Ⅱ | 5μl | | 水补足总体积 | 50μl |
|
② 25°C 保温30分钟，再 37°C 保温16小时；
③ 65°C ，20分钟可以热失活蛋白脱糖混合液 II。
变性酶切
| 组分 | 体积 |
|---|
| 底物 | 10-100μg 糖蛋白（10-20μl）
| | 10×糖蛋白变性缓冲液 | 2.5 μl | 水补足总体积
| 25 μl
| | （100℃孵育10 min，冷却至室温后加入以下试剂） |
| | 10×糖苷酶切反应液 | 5 μl | | 10% NP-40 | 5 μl
| | 蛋白脱糖混合液Ⅱ | 5 μl
| | 水补足总体积 | 50μl |
|
注意事项
-胎球蛋白可作为糖蛋白的阳性参照物；
-不建议用于粘蛋白样糖蛋白脱糖；
-本品收到后尽量避免冻融；
-使用时请穿实验服并佩戴一次性手套；
-本品不得直接用于临床诊断和治疗。
Expression system: E.coli
Unit definition:
PNGase F: One unit is defined as the amount of enzyme required to remove >95% of the carbohydrates from 10 μg of denatured RNase B in a 10‑μL reaction system at 37 °C for 1 hour.
O‑Glycosidase: One unit is defined as the amount of enzyme required to remove 0.68 nmol of O‑linked glycans from 5 mg of sialidase‑treated, non‑denatured fetuin in a 100‑μL reaction system at 37 °C for 1 hour.
Sialidase (α2‑3,6,8,9): One unit is defined as the amount of enzyme required to cleave >95% of terminal α‑Neu5Ac from 1 nmol of Neu5Acα2‑3Galβ1‑3GlcNAcβ1‑3Galβ1‑4Glc‑7‑amino‑4‑methyl‑coumarin (AMC) in a total reaction volume of 10 μL at 37 °C for 5 minutes.
β1‑4 Galactosidase: One unit is defined as the amount of enzyme required to hydrolyze >95% of the terminal β‑D‑mannose from 1 nmol of Galβ1‑4GlcNAcβ1‑3Galβ1‑4Glc‑7‑amino‑4‑methyl‑coumarin in a 10‑μL reaction system at 37 °C for 1 hour.
β‑N‑Acetylhexosaminidase: One unit is defined as the amount of enzyme required to hydrolyze >95% of terminal β‑D‑N‑acetylgalactosamine from 1 nmol of GalNAcβ1‑4Galβ1‑4Glc‑7‑amino‑4‑methyl‑coumarin (AMC) in a 10‑μL reaction system at 37 °C for 1 hour.
This product is a mixture of five recombinant enzymes expressed in E. coli, as detailed below:
① PNGase F: Derived from Elizabethkingia miricola, with a molecular weight of ~36 kDa. It selectively hydrolyzes all Asn‑linked N‑glycosidic bonds of proteins except those carrying an α1‑3‑fucosidic linkage. It cleaves the glycosidic bond between asparagine (Asn) and the innermost GlcNAc, releasing asparagine‑linked glycans from glycoproteins and converting asparagine into aspartic acid. It processes high‑mannose, hybrid and complex‑type oligosaccharides.
② O‑Glycosidase (Endo‑α‑N‑acetylgalactosaminidase): Derived from Enterococcus faecalis, molecular weight ~147 kDa. After sialidase removes terminal sialic acids from glycoproteins, O‑glycosidase catalyzes removal of core‑1 and core‑3 O‑linked disaccharide structures on glycoproteins.
③ Sialidase (α2‑3,6,8,9): Derived from Arthrobacter ureafaciens, molecular weight ~100 kDa. This enzyme hydrolyzes sialic acids linked via α2‑3, α2‑6, α2‑8 and α2‑9 linkages on glycoproteins and oligosaccharides. Hydrolysis rates for α2‑3 and α2‑6 linkages are higher than for α2‑8 and α2‑9 linkages.
④ β1‑4 Galactosidase: Derived from Streptococcus pneumoniae, molecular weight ~250 kDa. A highly‑specific exoglycosidase that catalyzes hydrolysis of non‑reducing terminal galactose residues on oligosaccharides.
⑤ β‑N‑Acetylhexosaminidase: Derived from Streptomyces plicatus, molecular weight ~100 kDa. It catalyzes hydrolysis of terminal β‑D‑N‑acetylgalactosamine and glucosamine residues on oligosaccharides, and can remove O‑GlcNAc (N‑acetylglucosamine linked to serine/threonine).
Synergistic action of the five enzymes removes the vast majority of N‑linked, simple O‑linked, and certain complex O‑linked glycan chains from glycoproteins.
Enzyme Activity Unit Definition
① PNGase F: One unit is defined as the amount of enzyme required to remove >95 % of carbohydrate from 10 μg denatured RNase B in a 10 μL reaction incubated at 37 °C for 1 h.
② O‑Glycosidase: One unit is defined as the amount of enzyme required to remove 0.68 nmol O‑linked glycans from 5 mg sialidase‑treated native fetuin in a 100 μL reaction incubated at 37 °C for 1 h.
③ Sialidase (α2‑3,6,8,9): One unit is defined as the amount of enzyme required to cleave >95 % of terminal α‑Neu5Ac from 1 mmol Neu5Acα2‑3Galβ1‑3GlcNAcβ1‑3Galβ1‑4Glc‑7‑amino‑4‑methyl‑coumarin (AMC) in a total reaction volume of 10 μL incubated at 37 °C for 5 min.
④ β1‑4 Galactosidase: One unit is defined as the amount of enzyme required to hydrolyze >95 % of terminal β‑D‑galactose (note: original text typo “β‑D‑mannose”) from 1 mmol Galβ1‑4GlcNAcβ1‑3Galβ1‑4Glc‑7‑amino‑4‑methyl‑coumarin in a 10 μL reaction incubated at 37 °C for 1 h.
⑤ β‑N‑Acetylhexosaminidase: One unit is defined as the amount of enzyme required to hydrolyze >95 % of terminal β‑D‑N‑acetylgalactosamine from 1 mmol GalNAcβ1‑4Galβ1‑4Glc‑7‑amino‑4‑methyl‑coumarin (AMC) in a 10 μL reaction incubated at 37 °C for 1 h.
Content per Vial
| Enzyme | 5 rxns | 20 rxns |
|---|
| PNGase F | 2.5 KU/vial | 10 KU/vial | | O‑Glycosidase | 20 KU/vial | 80 KU/vial | | α2‑3,6,8,9 Sialidase | 100 U/vial | 400 U/vial | | β1‑4 Galactosidase | 250 U/vial | 1000 U/vial | | β‑N‑Acetylhexosaminidase | 75 U/vial | 300 U/vial |
|
Product Contents
| Storage Unit No. | Storage Unit Name | Component No. | Component Name | Appearance | 5rxns |
| 20rxns | Storage | | P1527889WT | Protein Deglycosylation Mix II(2-8℃ Components) | P1527889A | Protein Deglycosylation Mix II | Liquid | 25µl |
| 100µl | 2-8℃. | | P1527889DT | Protein Deglycosylation Mix II(-20℃ Components) | P1527889B | 10× Glycosidase Cleavage Reaction Buffer | Liquid | 200µl |
| 200µl | -20℃ |
|
| P1527889C | 10% NP-40 | Liquid | 200μl |
| 200μl | -20℃ |
|
| P1527889D | Fetuin‑A (8mg/ml) | Liquid | 50μl |
| 50μl | -20℃ |
|
| P1527889E | 10× Glycoprotein Denaturing Buffer | Liquid | 200μl |
| 200μl | -20℃ |
Note: During shipment, each component is individually packaged and transported under temperaturecontrolled conditions corresponding to its storage requirement ( 2-8°C/-20°C ) to maintain optimal product performance. For a single order containing components requiring different storage temperatures, packages may be delivered in separate shipments, subject to actual receipt. Upon receipt, store all components immediately according to the temperature specifications listed in the component list. The shelflife of all unopened components is 12 months from the date of manufacture, provided that both shipping and storage conditions are strictly followed. |
Product Applications
Removal of the vast majority of N‑linked, simple O‑linked, and certain complex O‑linked glycan chains from glycoproteins.
Protocol (For Reference Only)
Native Digestion
Prepare reaction system as below:
| Component | Volume |
|---|
| Substrate | 10‑100 μg glycoprotein (10‑40 μL) | | 10× Glycosidase Digestion Buffer | 5 μL | | Protein Deglycosylation Mix II | 5 μL | | Water | Adjust total volume to 50 μL |
|
- Incubate at 25 °C for 30 min, then incubate at 37 °C for 16 h.
- Heat‑inactivate Protein Deglycosylation Mix II at 65 °C for 20 min.
Denaturing Digestion
| Component | Volume |
|---|
| Substrate | 10‑100 μg glycoprotein (10‑20 μL) | | 10× Glycoprotein Denaturation Buffer | 2.5 μL | | Water | Adjust total volume to 25 μL |
|
Incubate at 100 °C for 10 min, cool to room temperature, then add reagents below:
| Component | Volume |
|---|
| 10× Glycosidase Digestion Buffer | 5 μL | | 10 % NP‑40 | 5 μL | | Protein Deglycosylation Mix II | 5 μL | | Water | Adjust total volume to 50 μL |
|
Notes
‑ Fetuin A may be used as positive control for glycoprotein deglycosylation.
‑ Not recommended for deglycosylation of mucin‑like glycoproteins.
‑ Avoid repeated freeze‑thaw cycles upon receipt.
‑ Wear lab coat and disposable gloves when handling.
‑ Not intended for direct clinical diagnosis or therapeutic use.
危险品化学品经营许可证（带存储）